preclinical pharmacokinetics and tissue distribution of long-acting

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    Preclinical Pharmacokinetics and Tissue Distribution of Long-Acting 1

    Nanoformulated Antiretroviral Therapy 2

    Nagsen Gautam1, Upal Roy2, Shantanu Balkundi2, Pavan Puligujja2, Dongwei Guo2, 3

    Nathan Smith2, Xin-Ming Liu2, Benjamin Lamberty2, Brenda Morsey2, Howard S. Fox2, 4

    JoEllyn McMillan2, Howard E. Gendelman2, and Yazen Alnouti1 # 5


    1 Department of Pharmaceutical Sciences, College of Pharmacy, University of 7

    Nebraska Medical Center, Omaha, NE 68198-6025, USA 8

    2 Departments of Pharmacology and Experimental Neuroscience and, University of 9

    Nebraska Medical Center, Omaha, NE 68198-5215, USA 10

    Running title: Pharmacokinetics of nanoformulated antiretrovirals 11


    # Corresponding author: 13

    Yazen Alnout i 14

    Department of Pharmaceutical Sciences, College of Pharmacy 15

    University of Nebraska Medical Center 16

    986025 Nebraska Medical Center, Omaha, NE 68198-6025 17

    Phone: 402-559-4631 18

    Fax: 402-559-9543 19

    E mail: 20

    21 22

    Copyright 2013, American Society for Microbiology. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.00267-13 AAC Accepts, published online ahead of print on 22 April 2013

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    Abstract 23

    Long-acting injectable nanoformulated antiretroviral therapy (nanoART) was 24

    developed with the explicit goal of improving medicine compliance and for drug 25

    targeting of viral tissue reservoirs. Prior nanoART studies completed in humanized 26

    virus-infected mice demonstrated sustained antiretroviral responses. However, the 27

    pharmacokinetic (PK) and tissue distribution of nanoART were not characterized. To 28

    this end, PK and tissue distribution of nanformulated atazanavir (ATV) and ritonavir 29

    (RTV) injected subcutaneously or intramuscularly in mice and monkeys were 30

    evaluated. Fourteen days after injection, ATV and RTV levels were up to 13-, 41- and 31

    4500- fold higher than those resulting from native drug administration in plasma, 32

    tissues, and at the site of injection, respectively. At 10, 50, 100, and 250 mg/kg 33

    nanoART doses, more and less than proportional increases in plasma and tissue levels 34

    with dose relationships were demonstrated with ATV and RTV. Multiple-dose 35

    regimens showed serum and tissue concentrations up to 270-fold higher than native 36

    drug throughout eight-weeks of study. Importantly, nanoART was localized in non-37

    lysosomal compartments in tissue macrophages creating intracellular depot sites. 38

    Reflective data were obtained in representative rhesus macaque studies. We conclude 39

    that nanoART demonstrates enhanced blood and tissue antiretroviral drug levels over 40

    native drugs. The sustained and enhanced PK profile of nanoART, at least in part, is 41

    the result of the sustained release of ATV and RTV from tissue macrophases and at 42

    the site of injection. 43



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    Introduction 46

    The development of effective antiretroviral therapy (ART) has transformed 47

    human immunodeficiency virus (HIV) disease into a long-term and manageable 48

    disorder (1). Infected patients can live well past their fifth, sixth and seventh decades 49

    of life (2). The shortcomings for ART are, notably, associated with viral resistance and 50

    rebounds that may occur despite long periods of undetectable virus in blood (3). This 51

    can occur despite the presence of therapeutic plasma drug levels, which is attributed to 52

    activation of latent virus hidden in anatomical and intracellular reservoirs (4). As ART 53

    regimens are commonly ineffective in reaching viral sanctuaries, viral replication occurs 54

    continuously at low levels as a result of ineffective antiretroviral penetrance into viral 55

    reservoirs including the lymphoid and central nervous system reservoirs (5), which 56

    allows HIV to circumvent eradication (6, 7). Thus, life-long treatment is needed in 57

    order to suppress the virus and to enable the patient to remain clinically asymptomatic 58

    (8). 59

    Patients compliance with medications intake plays yet another crucial role for 60

    disease management (9), which becomes even more challenging with complex ART 61

    regimens that require long-term adherence for often substantive pill burdens (10). 62

    Drug-regimen compliance has also been identified as a critical risk factor for viral 63

    resistance (11, 12). In attempts to overcome such limitations, long-acting 64

    nanoformulated ART (nanoART) was developed to achieve steady state drug levels 65

    with infrequent dosing (13, 14). Moreover, as HIV-infected individuals serve as 66

    vehicles for viral transmission, long acting formulations could also serve to decrease 67

    viral spread (15, 16). As ART leads to spectrum of toxicities and drug-drug interactions 68

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    causing added disease morbidities, these may be overcome by improved viral 69

    suppression and reduced toxicities through nanoART (17). 70

    While previous reports demonstrated that mononuclear phagocytes (MP; 71

    monocytes and macrophages) can act as reservoirs and transporters of HIV-1, these 72

    cells could also potentially serve to facilitate drug uptake, transport, and release of 73

    nanoART (18-21). Using MP cell culture systems, it was shown that uptake and 74

    release of nanoART into and from monocyte-derived macrophages (MDM) are 75

    sustained at levels equal to or beyond the effective concentrations (EC50) with limited 76

    cytotoxicity (22). This was achieved after optimizing the shape, size, and charge of the 77

    nanoparticles for cell entry and release of two commonly administered protease 78

    inhibitors, atazanavir (ATV) and ritonavir (RTV) (23, 24). After passing in vitro 79

    screening, ATV and RTV nanoformulations were selected for in vivo pharmacokinetic 80

    (PK) studies (13). While the pilot efficacy studies in humanized virus-infected mice 81

    demonstrated effective and sustained antiretroviral responses, dosing regimens and 82

    tissue and cell biodistributions remained incomplete (13, 14, 25). To these ends, we 83

    characterized the pharmacokinetics and biodistribution of nanoART in mice and in 84

    monkeys after subcutaneous (SC) administration. Results showed clear improvements 85

    in the PK profile over native (unformulated) drug at various dosing regimens. 86

    Intracellular nanoART reservoirs associated with endosomal MP compartments (23) in 87

    tissues paralleled what had previously been demonstrated in in vitro studies (18) and 88

    resulted in sustained and enhanced systemic drug levels in vivo. These preclinical 89

    studies may further enable the development of nanoART for clinical intervention (26, 90

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    27). Such formulations would provide advantages in PK properties and patient 91

    compliance over what is now established by conventional native drug regimens (9). 92

    Materials and methods 93 94 Chemicals 95

    Free base RTV was obtained from Shengda Pharmaceutical Co. (Zhejiang, 96

    China). ATV-sulfate was purchased from Gyma Laboratories of America Inc. 97

    (Westbury, NY, USA). Lopinavir (LPV) was purchased from Toronto Research 98

    Chemicals Inc. (North York, Ontario, Canada). HPLC-grade methanol, acetonitrile, 99

    ammonium acetate, acetic acid, propylene glycol and phosphate buffered saline- 1X 100

    were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Isoflurane was obtained 101

    from Halocarbon Product Corporation (River Edge, NJ, USA). BD- 28G-1/2 insulin 102

    syringes were obtained from Becton Dickinson and Company (Franklin Lakes, NJ, 103

    USA). Cremophore EL and poloxamer 188 (P188) were obtained from Sigma-Aldrich 104

    (St. Louis, MO, USA) and ethyl alcohol from Acros Organics (NJ, USA) 105

    Preparation and characterization of nanoART 106

    NanoART RTV and ATV were prepared with polymer excipients by high-107

    pressure homogenization as described previously (28). These formulations consisted 108

    of crystalline drug surrounded by a thin layer of a P188 surfactant (29). Drug loading 109

    was analyzed by high performance liquid chromatography (HPLC-UV) [11] and by 110

    ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) 111

    (30). These formulations were screened for cell uptake, retention, release and 112

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    antiretroviral activity using human monocyte-derived macrophages as described 113

    previously (18, 22, 28). 114

    Mouse studies 115

    Eight-week-old, healthy male Balb/C mice were purchased from Charles River 116

    Laboratories (Wilmington, MA). Sterilized 7012 Teklad diets (Harlan, Madison, WI) 117

    were used for mice, and water was provided ad libitum. Mice were housed in the 118

    University of Nebraska Medical Center (UNMC)